With the development of single molecule imaging technology, researchers can observe the extension of a single base in the DNA/RNA chain, which greatly facilitates the development of the third generation gene sequencing technology-single molecule sequencing technology. By adding one base at each time to a complementary sequence of the object to be tested, the complementary strand is continuously extended, and finally we will obtain the entire gene sequence of the strand to be tested. Compared with the current popular second generation gene sequencing technologies, single molecule sequencing technology does not require polymerase chain reaction (PCR) amplification, therefore reducing the sequencing error rate.
In order to achieve single-molecule sequencing, many methods have been tried, such as column separation, radioisotope labeling, and the like. However, these methods do not have the resolution required for single molecule sequencing. Fluorescence microscopy has become the primary method of single molecule sequencing due to its higher resolution, lower cost and simplicity of operation. There are three common methods of fluorescence microscopy: epi-fluorescence microscopy, confocal fluorescence microscopy and total internal reflection fluorescence (TIRF) microscopy. Resolution of the confocal and TIRF imaging is higher than that of epi-fluoresence imaging, while confocal imaging requires a long time to scan for forming an image, such that it is relatively not suitable for single molecule sequencing. The light path and light path adjustment of the current total internal reflection fluorescence microscopes need improvement.